The mammalian protein kinase activity stimulated preferentially by low concentrations of guanosine 3':5'-monophosphate (cyclic GMP), but not by adenosine 3':5'-monophosphate (cyclic AMP), was readily assayed in a modified incubation system that contained a neutral phosphate buffer, protein kinase modulator, and arginine-rich histone. Cyclic GMP-dependent protein kinase activity assayed under these conditions was about two to three orders of magnitude higher than that previously detected. The enzyme activity occurred in all guinea pig and rat tissues (lung, heart, aorta, brain, liver, ileum, adipose, and pancreatic islets) examined. The activity can be separated from the cyclic AMP-dependent protein kinase activity, also present in the same tissues, by means of either Sephadex G-200 gel filtration or ammonium sulfate fractionation. The cyclic GMP-dependent efzyme preparations had Ka values for cyclic GMP ranging form 0.03 to 0.12 micron M, compared to the Ka values for cyclic AMP ranging from 0.6 to 3.8 micron M. The presence of phosphate and protein kinase modulator was essential for maximal cyclic GMP-dependent enzymy activity. The occurrence of high levels of cyclic GMP-dependent protein kinase activity in mammalian tissues clearly suggests that it may serve as a "target" enzyme for cyclic GMP, mediating many biological effects of this cyclic nucleotide.